Method for predicting cardiotoxicity risk in cancer patients receiving anthracyclines chemotherapy

ABSTRACT

The present invention refers to an in vitro method for predicting cardiotoxicity risk in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy based on the determination of the expression levels of a combination of 10 circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, in a biological sample isolated from the patient. The present invention also refers to said set of 10 circulating miRNAs for its use as biomarker of prediction of cardiotoxicity risk in cancer patients receiving, or susceptible to receive, anthracyclines chemotherapy. Finally, a method for the prevention of cardiotoxicity, in patients receiving or susceptible to receive anthracyclines chemotherapy, that comprises modulating the expression levels of the set of said 10 circulating miRNAS is contemplated.

FIELD OF THE INVENTION

The present invention relates generally to the field of Oncology and Toxicology. Particularly, the invention relates to a method for predicting cardiotoxicity risk in cancer patients receiving, or susceptible to receive, anthracyclines chemotherapy. The method is based on the analysis of the expression levels of a set of 10 circulating miRNAs.

BACKGROUND OF THE INVENTION

Anthracyclines are well-established and effective anti-neoplastic drugs currently used alone or in combination with other anti-mitotic agents for the treatment of a wide variety of tumors including breast cancer (Palmieri C, Krell J, James C R, et al. Rechallenging with anthracyclines and taxanes in metastatic breast cancer. Nat Rev Clin Oncol 2010; 7(10): 561-741). However, its implementation is related with several collateral effects, being cardiotoxicity the most severe. Usually the first symptoms appear within the first year of administration of the last dose, but it may occur even after 6-10 years after administration (Cardinale D, Colombo A, Bacchiani G, et al. Early detection of anthracycline cardiotoxicity and improvement with heart failure therapy. Circulation 2015; 131(22): 1981-82).

Anthracyclines induced cardiotoxicity (AC) is considered acute if occurs shortly after its administration (Cardinale D. et al; Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis 2007; 49(5): 330-52). However, since the toxicity of anthracyclines is cumulative, the most common type of AC occurs several years after the treatment what is known as chronic cardiotoxicity. Since the heart is a post-mitotic organ, once the myocardium is damaged the deterioration of heart function is commonly progressive. Early assessment for cardiac toxicity following anthracycline treatment could aid to perform a more precise risk stratification of patients who may benefit from more frequent surveillance cardiac function.

miRNAs have been involved in pivot biological processes such as drug resistance (Ahmad N, Haider S, Jagannathan S, Anaissie E, Driscoll J J. MicroRNA theragnostics for the clinical management of multiple myeloma. Leukemia 2014; 28(4): 732-8), cellular damage or cancer (Allegra A, Alonci A, Campo S, et al. Circulating microRNAs: new biomarkers in diagnosis, prognosis and treatment of cancer (review). Int J Oncol 2012; 41(6): 1897-912). These molecules are a small class of single trans noncoding RNAs between 20-25 nucleotides that control expression of mRNAs at the post-transcriptional level by targeting the 3″untranslated region of mRNA transcripts (Ambros V. The functions of animal microRNAs. Nature 2004; 431(7006): 350-5). They can cleave complementary messenger mRNA targets and diminish the translation of partially complementary targets (Kim V N. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 2005; 6(5): 376-85). These short molecules have high specificity and many of them have a tissue specific expression (Latronico M V, Catalucci D, Condorelli G. Emerging role of microRNAs in cardiovascular biology. Circ Res 2007; 101(12): 1225-36). Due to its high stability in plasma and serum and easy detection they have been used as biomarkers in different pathologies including cancer (Hamam R, Hamam D, Alsaleh K A, et al. Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers. Cell Death Dis 2017; 8(9): e3045) and cardiac diseases (Gilad S, Meiri E, Yogev Y, et al. Serum microRNAs are promising novel biomarkers. PLoS One 2008; 3(9): e3148; Mitchell P S, Parkin R K, Kroh E M, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008; 105(30): 10513-8; Schulte C, Zeller T. microRNA-based diagnostics and therapy in cardiovascular disease-Summing up the facts. Cardiovasc Diagn Ther 2015; 5(1): 17-36) and specific patterns of circulating miRNAs have been associated with heart failure (Tijsen A J, Creemers E E, Moerland P D, et al. MiR423-5p as a circulating biomarker for heart failure. Circ Res 2010; 106(6): 1035-9) and myocardial infarction (Wang G K, Zhu J Q, Zhang J T, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010; 31(6): 659-66). This circulating miRNAs were found stable under unfavourable conditions as high or low PH, boiling and multiple freeze-thaw cycles (Mitchell P S, Parkin R K, Kroh E M, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008; 105(30): 10513-8; Lawrie C H, Gal S, Dunlop H M, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 2008; 141(5): 672-5) suggesting that circulating miRNAs are protected from degradation.

Currently there are few studies linking treatment with anthracyclines and the detection of circulating microRNAs. Using induced pluripotent stem cells (iPSC) derived cardiomyocytes (CM) treated with Doxorubicin it was shown a deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303 in treated cells in comparison to non-treated controls (Chaudhari U, Nemade H, Gaspar J A, Hescheler J, Hengstler J G, Sachinidis A. MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes. Arch Toxicol 2016; 90(12): 3087-98). In vivo, it was reported the usefulness of miR-208 (Calvano J, Achanzar W, Murphy B, et al. Evaluation of microRNAs-208 and 133a/b as differential biomarkers of acute cardiac and skeletal muscle toxicity in rats. Toxicol Appl Pharmacol 2015; Nishimura Y, Kondo C, Morikawa Y, et al. Plasma miR-208 as a useful biomarker for drug-induced cardiotoxicity in rats. J Appl Toxicol 2015; 35(2): 173-80), miR-133a/b, miR146a (Horie T, Ono K, Nishi H, et al. Acute doxorubicin cardiotoxicity is associated with miR-146a-induced inhibition of the neuregulin-ErbB pathway. Cardiovasc Res 2010; 87(4): 656-64) and miR-34a (Desai V G, J C K, Vijay V, et al. Early biomarkers of doxorubicin-induced heart injury in a mouse model. Toxicol Appl Pharmacol 2014; 281(2): 221-9) as useful biomarkers for drug-induced cardiotoxicity in rats. Recently, the levels of several miRNAs were analyzed in blood of childhood patients treated with anthracyclines and determined that only miR-29b and miR-499 significantly correlated with cumulative anthracycline dosage shortly after infusion (Leger K J, Leonard D, Nielson D, de Lemos J A, Mammen P P, Winick N J. Circulating microRNAs: Potential Markers of Cardiotoxicity in Children and Young Adults Treated With Anthracycline Chemotherapy. J Am Heart Assoc 2017; 6(4)). In this context, some molecular prognostic classifiers based on miRNA detection have been developed for cardiac induced toxicity after treatment with Trastuzumab (Ezaz G, Long J B, Gross C P, Chen J. Risk prediction model for heart failure and cardiomyopathy after adjuvant trastuzumab therapy for breast cancer. J Am Heart Assoc 2014; 3(1): e000472) or anthracyclines (Leger K J, Leonard D, Nielson D, de Lemos J A, Mammen P P, Winick N J. Circulating microRNAs: Potential Markers of Cardiotoxicity in Children and Young Adults Treated With Anthracycline Chemotherapy. J Am Heart Assoc 2017; 6(4); Leger K, Slone T, Lemler M, et al. Subclinical cardiotoxicity in childhood cancer survivors exposed to very low dose anthracycline therapy. Pediatr Blood Cancer 2015; 62(1): 123-7; Lipshultz S E, Diamond M B, Franco V I, et al. Managing chemotherapy-related cardiotoxicity in survivors of childhood cancers. Paediatr Drugs 2014; 16(5): 373-89). However, none of them can be used for personalized risk assessment before the initiation of chemotherapy.

The present invention provides a novel predictive model based on the combination of ten selected circulating miRNAs that can be used to stratify cancer patients at risk of cardiotoxicity.

The definition of this microRNA signature for cardiotoxic risk in cancer patients can be used for decision making of treatment regimens together with the monitorization of cardiotoxicity in cancer patients treated with anthracyclines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Venn diagram of the overlap of miRNA profiles differentially expressed between cases and controls in miRNAseq array and list of the 10 miRNAs.

FIG. 2 (A, B). Graphics showing relative values of the 10 miRNAs (miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p) in controls and cases (N=100) as detected by qPCR. Results are expressed as mean±SEM. A.U. stands for arbitrary units. *P<0.05.

FIG. 3. (A) Determination of probability of developing cardiotoxicity (positive result) using a beta mixed model assessing the association between left ventricular ejection fraction (LVEF) and the miRNA included in MirCaTox. (B) Area under the ROC curve (AUC) showing sensitivity and specificity of the model.

FIG. 4. (A, B) Graphics showing modulation of miRNAs by Doxorubicin in human cardiomyocytes and patients' serum. Left panels show copy number of indicated miRNAs in cardiomyocytes in culture before or after the addition of Doxorubicin (1 μM) for 24 h. Right panels show copy numbers of indicated miRNAs as detected by RNAseq in serum from control patients and cases and expressed as mean±SEM (N=10 in each case) before or after completion of anthracycline chemotherapy. Only miRNAs showing a similar tendency in cells and patients' serum are illustrated. *P<0.05.

FIG. 5 (I-J). Target genes of the 10 miRNAs (miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p).

FIG. 6. Cardioprotective effects induced by overexpression of miR-30b-5p, miR-150-5p and miR-4732-3p in neonatal rat cardiomyocyte cultures (NRCMs) treated with Doxorubicin (1 μM) for 24 h in terms of cell viability (CCK8 assay) and percentage of apoptotic cells (Anexin-V positive cells). Results are expressed as mean±SEM. Cell viability is normalized to viability showed in cultures transduced with a negative control (NC) miRNA mimic and non-treated with Doxorubicin. *P<0.05, **P<0.01

FIG. 7. Reoxygenation after ischemia modulates expression of MIRCATOX miRNAs. Effect of ischemia followed by reperfusion (I/R) in miRNA-4732-3p and miRNA-150-5p levels as quantified by qPCR. Absolute copy numbers of miRNAs are indicated in cardiomyocyte cultures treated or not with I/R culture conditions. Results are expressed as mean±SEM. *P<0.05.

FIG. 8. Cardioprotective effects induced by overexpression of miR-150-5p and miR-4732-3p in neonatal rat cardiomyocyte cultures (NRCMs) subjected to 7 h ischemia (defined as deprivation of oxygen and nutrients) followed by 30 min reperfusion (reoxygenation and nutrient suplementation) in terms of decrease of reactive oxygen species (ROS) and apoptosis (Anexin V) in NRCMs. Results are expressed as mean±SEM. A.U. stands for arbitrary units. *P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides with new biomarkers of prediction of cardiotoxicity risk in cancer patients receiving, or susceptible to receive, anthracyclines chemotherapy.

In particular, the authors have developed a microRNA signature consisting of 10 circulating miRNAs: miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, that allows the identification of patients with high risk to suffer a decline of cardiac function after anthracyclines treatment.

The alteration of the expression of these miRNAs, comparing with the expression of said miRNAs in a control subject, not suffering from cardiotoxicity, is indicative of cardiotoxicity risk.

In the present invention, a control subject is a cancer patient not suffering from cardiotoxicity after anthracycline treatment.

For the purposes of the present invention, cardiotoxicity is defined, according to the most recent clinical guidelines, as the presence, in transthoracic echocardiography performed by a qualified physician, of one of the following situations:

-   -   1. A reduction of Left Ventricle Ejection Fraction (LVEF) >5% to         a final LVEF <55%, with symptoms of heart failure, or     -   2. A reduction of LVEF >10% to a final LVEF <55, without the         presence of symptoms.

According to the above, in a main aspect, the present invention refers to the use of a set of 10 circulating miRNAs, consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, as biomarker of prediction of cardiotoxicity risk in cancer patients receiving, or susceptible to receive, anthracyclines chemotherapy.

This new miRNA signature has allowed the authors of the invention to develop a method for predicting cardiotoxicity risk in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy. This method comprises the following steps:

-   -   i. Determining the expression levels of a combination of 10         circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA         30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p,         miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and         miRNA-4732-3p, in a biological sample isolated from the patient,         and     -   ii. Comparing the expression levels determined in i) with the         expression levels of said miRNAs from a biological sample         isolated from a control subject, wherein the alteration of the         expression of these miRNAs, comparing with the expression of         said miRNAs in a control subject not suffering from         cardiotoxicity, is indicative of cardiotoxicity risk.

In addition, based on this miRNA signature, the authors of the present invention have developed methods that, linked to an algorithm, allow cardiotoxicity to be detected with a probabilistic value, which is very useful in making clinical decisions. These methods can be used to screen cardiotoxic risk in patient's cohort receiving anthracyclines treatment or that are going to be subjected to anthracyclines chemotherapy and be used as powerful tools for cardiotoxic risk stratification of cancer patients.

Preferably, the probability of suffering from cardiotoxicity in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy is calculated according to the following formula:

${\Pr({Cardiotoxicity})} = \frac{e^{LP}}{1 + e^{LP}}$

-   -   wherein,     -   LP=−1.228−0.041*miR4732−0.066*miR22−0.02*miR30b+0.081*miR16−0.053*miR148a+0.012*miR192−0.009*miR150p−0.055*miR215+0.0899*miR486

Therefore, in another aspect, the present invention refers to an in vitro method for predicting the cardiotoxicity risk probability in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy comprising:

-   -   a) Determining the expression levels of a combination of 10         circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA         30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p,         miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and         miRNA-4732-3p, in a biological sample isolated from the patient,         and     -   b) Introducing the expression data obtained in a) in the         following equation:

${\Pr({Cardiotoxicity})} = \frac{e^{LP}}{1 + e^{LP}}$

-   -   wherein,     -   LP=−1.228−0.041*miR4732−0.066*miR22−0.02*miR30b+0.081*miR16−0.053*miR148a+0.012*miR192−0.009*miR150p−0.055*miR215+0.0899*miR486

The obtained result (Pr(cardiotoxicity) is the expected probability of cardiotoxicity.

The miR92b-3p is used intrinsically in the equation, since it was the miRNA that was used to normalize the values of the rest of miRNAs obtained by qPCR. This miRNA is appropriate for the normalization, according to qPCR method specifications for miRNAs amplification.

The combination of the 10 miRNAs is valid to predict cardiotoxicity risk both before or once the chemotherapy has been initiated.

For the purposes of the invention, the determination of the miRNAs expression levels could be carried out by different techniques of Molecular Biology, for example qPCR or any other alternative technique generally used in the state of the art.

In a preferred embodiment, the biological sample isolated from the sample is a liquid biopsy (serum, plasma, urine, blood, etc). In a more preferred embodiment, the liquid biopsy is serum.

In a particular embodiment, the patient receiving, or susceptible to receive, anthracyclines chemotherapy is a breast cancer patient.

The modulation of the expression of the miRNAs included in the miRNA signature of the present invention result in a cardioprotection effect of cardiomyocyte cells. Accordingly, in another main aspect, the present invention refers to a method for the prevention of cardiotoxicity, in patients receiving or susceptible to receive anthracyclines chemotherapy, that comprises modulating the expression levels of the set of 10 circulating miRNAS consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p.

EXAMPLES

Materials and Methods

The study was carried out according to the principles of the Declaration of Helsinki.

The researchers assured that the privacy of the patients is guaranteed. All procedures were approved by local and national ethical committees. Written informed consent was obtained from each patient.

Study Design

Prospective Observational Study

Study Population

Breast cancer patients post- or pre-antineoplastic treatment, >18 years old at time of inclusion.

Main Study Parameters/Endpoints

The primary endpoint is the development of cardiotoxicity, defined as the most recent clinical guidelines: The presence, in transthoracic echocardiography performed by a qualified physician of one of the following situations:

-   -   1. A reduction of Left Ventricle Ejection Fraction (LVEF) >5% to         a final LVEF <55%, with symptoms of heart failure.     -   or     -   2. A reduction of LVEF >10% to a final LVEF <55, without the         presence of symptoms.

Patients

The study population included 137 patients with different types of breast cancer that were going to receive anthracyclines. The number of positive cases of acute cardiotoxicity was 10.2%. The period for inclusion of patients was 3 years. The follow-up duration was 2 years.

The study population consisted of HER2-negative and HER2-positive breast cancer patients (n=137). These patients were scheduled to undergo three different adjuvant therapies as indicated:

-   -   1) TAC chemotherapy consisted in Docetaxel 75 mg/m2, Doxorubicin         (Adriamycin) 50 mg/m² and Cyclophosphamide 500 mg/m², cycled         every 21 day for 6 cycles (N=22).     -   2) AC chemotherapy consisted in Doxorubicin (adriamycin) 60         mg/m² and Cyclophosphamide 600 mg/m2 cycled every 21 days for 4         cycles and then Paclitaxel (80 mg/m²) or Docetaxel (100 mg/m2)         weekly for 12 weeks or cycled every 21 days for 4 cycles         respectively (N=90).     -   3) CAELIX chemotherapy, the same treatment that AC chemotherapy         but with Liposomal doxorubicin (Caelix) instead Doxorubicin         (adriamycin) (N=15).     -   4) FEC (N=4)     -   5) Taxanes (N=7)

Clinical Data Collection

Clinical data was collected before and after receiving antineoplastic treatment. Data include demographics, medical history, cardiovascular risk factors, use of concomitant medication and/or antineoplastic regimens, symptoms and signs of cardiovascular disease, results of physical examination, echocardiography and routine laboratory results.

Serum Sample Collection

Blood samples were taken before chemotherapy directly before infusion and after completion of treatment in non-heparinized tubes. After two hours clotting, serum was harvested and stored at the Biobank of Hospital La Fe.

RNA Extraction

miRNA isolation was performed used miRNeasy kit (Qiagen Inc) according to the manufacture's instruction. Serum samples were thawed on ice and centrifuged for 10 min at 10,000 g 4° C. The supernatant of serum was added to QIAzol lysis reagent containing MS2 RNA and a known number of copies of cel-miR-39 as internal control. After vortex the mix, chloroform was added, and samples were incubated at room temperature for 5 min. Tubes were centrifuged for 15 min at 12,000 g at 4° C. 420 ul from the aqueous phase obtained after centrifugation was mixed with 280 μl of 100% ethanol. This mixture was loaded into an RNeasy minElute spin column in a 2 mL collection tube and followed by centrifugation. The column was sequentially washed with Buffer RWT (700 μl) and Buffer RPE (500 μl) twice. The miRNA was eluted with 35 μl of RNase-free water.

Reverse Transcription

Reverse transcription (RT) was performed using miRCURY LNA™ Universal RT microRNA PCR Kit (Exiqon) following manufacturer's instructions. Briefly, 2 μl of RNA was mixed with 5× Reaction Buffer (2 μl), Enzyme mix (1 μl) and nuclease free water (5 μl) to a final volume of 10 μl. The mix was incubated for 60 min at 42° C. followed by transcriptase heat-inactivation by incubating 5 min at 95° C. The product was frozen down until used.

miRNA Quantification

The quantification was performed using miRCURY LNA™ Universal RT microRNA PCR Kit and following manufacturer's instructions. Briefly, the cDNA was diluted in a 1/80 proportion in nuclease free water. 4 μl of diluted sample was mixed with 5 μl of PCR Master Mix and 1 μl of PCR primer set to a final volume of 10 μl each well. The temperature cycle program used for amplification was: 95° C. for 10 min, followed by 40 cycles at 95° C. for 15 s and 60° C. for 1 min. Real-time quantitative PCR was performed using the ViiA™ 7 Real-time PCR System (Applied Biosystems, Carlsbad, USA). Real-time monitoring of the PCR reactions was performed with the QuantStudio Real-Time PCR Software and DataAssist v3.01 (Applied Biosystems). Standard curves were made in parallel using a synthetic sequence of each miRNA. Hsa-mir-92b expression level was used as house-keeping control.

NRCMs Isolation

Neonatal rat cardiomyocytes (NRCMs) were isolated from newborn rats of 0-2 days old. Briefly, the newborn rats were euthanized by decapitation, their bodies were rinsed in ethanol 70% and the hearts were extracted in a laminar flow cabin. Hearts were minced in chunks of 1 mm³ and then incubated with trypsin at 4° C. in agitation O/N. Next day the tissue fragments were incubated with a solution of collagenase II/DNase at 37° C. 30′ and then were filtered through a 40 μM cell strainer to collect the cells. Cells were preplated for 2 h to purify the cardiomyocytes and then plated at a density of 150000 cells/cm². The NRCMs were maintained in Dulbecco's Modified Eagle Medium (DMEM) High Glucose (Gibco-Invitrogen®) supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen®), ciprofloxacin (MERCK), sodium pyruvate (MERCK) and L-glutamine (Gibco-Invitrogen®).

Cell Transfection and DOX Treatment

NRCMs were transfected using Lipofectamine® 3000 (ThermoFisher) according with manufacturer's protocol. We prepared Lipofectamine® 3000 and the Mimic separately in OptiMem (ThermoFisher), then mixed and incubated 15′ at room temperature. Finally, we added the transfection complexes to the NRCMs and incubated for 24 h. We treated the NRCMs with doxorubicin for 24 h with Doxorubicin 1 μM after cell transfection.

Ischemia-Reperfusion Injury Model In Vitro

An oxygen and glucose deprivation (OGD) protocol was established in our laboratory to mimic I/R-induced injury in vitro. NRVMs were incubated in none glucose DMEM (Gibco®) supplied with 1% of antibiotic mixture containing P/St (Sigma-Aldrich®) and submitted to a 1% O₂ and 5% CO₂ environment in a humidified incubator at 37° C. for 7 h (Ischemia, I). Reperfusion (R) was induced by subsequent incubation of cells with full high glucose DMEM medium (Gibco®) in 21% O₂ and 5% CO₂ atmosphere at 37° C. for 1 h. Cells cultured in normoxia (Nx) with full high glucose DMEM medium were used as control.

Cell Viability Assay

In order to evaluate the viability of the NRCMs after the treatments, we used the Cell Counting Kit 8 (CCK8, MERCK) assay following the manufacturer's instructions. The optical density of the cultures was measured at 450 nm in each well 4 h after incubation with the CCK8 assay solution.

Annexin-V Staining

To determine the percentage of apoptotic cells in the NRCMs, we performed an Annexin-V staining using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen). After the treatments the NRCMs were stained with Annexin-V and then analyze by flow cytometry.

Reactive Oxygen Species (ROS) Measurement

Oxidative stress was measured using CelIROX® Orange Reagent (C10443, Molecular Probes). Briefly, NRVMs were submitted to I/R protocol as described before and incubated with CelIROX® Orange Reagent solution and Hoescht33342 (1:2000, Thermo Scientific®). Live cells were visualised by In Cell Analyzer 2200 (GE Healthcare, UK) and orange intensity per cell was quantified with IN CELL Developer Tool software.

Echocardiography

Echocardiography was performed before and after the complete chemotherapeutic regime, and at 12 and 24 months after the end of the treatment. Conventional echocardiography consisted of two dimensional, and Doppler blood flow measurements to assess left ventricle (LV) structure and global function. All measurements were taken in accordance with the current guidelines of the American Society of Echocardiography using the iE33 (Philips Medical Systems, Andover, Mass., USA) with transthoracic S5-1 and X5-1 broadband transducers (frequency=1-5 MHz). Digital echocardiographic data were acquired during passively held end-expiration for offline analysis using a dedicated software. Parasternal long axis view was used to measure septal and posterior wall thickness and LV systolic and diastolic diameters. Cross-sectional images were recorded from the apex, and end-diastolic and end-systolic areas and LV lengths were measured from the apical four-chamber (A4C) and two-chamber (A2C) views (using the modified biplane Simpson's method) for the calculation of ejection fraction. The mitral flow velocity was measured by pulsed wave Doppler obtained in A4C view, with the sample volume positioned at the tips of the mitral valve leaflets. Peak early diastolic velocity—E wave, peak late diastolic velocity—A wave and E/A were calculated. All tracings were made by a single observer at a centralized reading center who was blinded to all other clinical or biomarker data.

Interactome Analysis

Functional blocks from Go biological process are used in this analysis. The tables below show the count of functional classes enriched in each of the comparisons and using each of the two databases. A significance level of 0.05 was used (ie. functional blocks are called enriched if their corrected p-value is smaller than 0.05). Enriched functional blocks are classified into:

-   -   up-regulated in the first biological condition compared to the         second one     -   down-regulated in the first biological condition compared to the         second one, and     -   not enriched.

The case of the GO database is special in that the functions or blocks of genes are organized into a directed acyclic graph (DAG) structure (https://en.wikipedia.org/wiki/Directed_acyclic_graph). This makes many of the significantly enriched functions to be redundant among them. In the second table of results of the functional profiling using the GO database we display just the significant and non redundant terms (functions); That is, the more specific terms among the significant ones.

Statistical Analysis

Since the analysis of RNAseq data can be very sensitive to the modelling decisions taken at each step, three different methods were employed to search for differentially expressed genes between positive and negative patients: Robinson and Smyth exact negative binomial test, Random Forest test and Elastic net test. The results of this analyses produced the list of miRNAs able to predict risk to suffer cardiac dysfunction after completion of chemotherapy. To develop a predictive algorithm for cardiotoxicity risk, a logistic mixed effects model considering the different previously selected miRNA as potential fixed effects predictors and the individuals as a random factor was adjusted. Variable selection was performed using Akaike's Information Criterion (AIC), selecting the model with the lowest AIC. Performance of the final model was assessed by estimating the Area under the ROC curve (AUC) and also a bootstrapped optimism-corrected version of it. P values<0.05 were considered statistically significant.

All statistical analyses were performed using R (version 3.3.1) and R-packages randomForest (version 4.6-12), glmnet (version 2.0-10), NBPSeq (version 0.3.0), Ime4 (version 1.1-12) and rms (version 4.5-0).

Results

Cardiac function in terms of left ventricular ejection fraction (LVEF) in a cohort of 137 breast cancer patients receiving anthracycline chemotherapy were analyzed. Demographic data is shown in Table 1. From this cohort, 37 were discarded by uncomplete data or sample collection.

TABLE 1 Clinical characteristics of the study population Controls Cases Demographics Age (years)  54.75 ± 12.42  53.46 ± 15.71 (35-76) (37-69) Sex (% female) 85 (100) 15 (100) BMI 26.43 ± 5.16 26.09 ± 5.41 Medical History Hipertension (%) 16 (18.8%) 1 (6.6%) Diabetes mellitus (%) 6 (7.0%) 2 (13.3%) Dislipemy (%) 31 (36.4%) 3 (20.0%) Smoking (%) 13 (15.3%) 2 (20%) Histological subtype of cancer Triple Negative 14 (16.5%) 4 (26.5%) Luminal A 5 (5.9%) 1 (6.6%) Luminal B 9 (10.6%) 2 (13.3) HER2/Neu 5 (5.9%) 3 (20%) Medication and cumulative dose Taxanes  615 ± 285 — TAC 491.34 ± 13.19 465.33 ± 70.81 AC 390.52 ± 9.81n 366.40 ± 20.61

From the final cohort (n=100), hematological parameters and serum biomarkers were determined before (Pre), after the last cycle of chemotherapy (Post) and one year after completion of chemotherapy (Rev), and the results are shown in Tables 2 and 3.

TABLE 2 Hematologic parameters Pre Post Rev White Leuco- Con- 6.64 ± 2.14 5.39 ± 0.19 5.48 ± 0.68 blood cytes trol cells (10³/μl) Cases 6.36 ± 1.81 5.62 ± 1.04 5.53 ± 0.77 Neutro- Con- 3.85 ± 1.87  1.40 ± 0.06* 3.2 ± 0.8 phils trol (10³/μl) Cases 3.80 ± 1.60  1.29 ± 0.17* 3.29 ± 0.9  Lympho- Con- 2.09 ± 0.70 3.29 ± 0.18 1.93 ± 0.55 cytes trol (10³/μl) Cases 1.88 ± 0.71  3.41 ± 0.93* 1.57 ± 0.48 Red Erytro- Con- 4.49 ± 0.47 4.16 ± 0.05 4.29 ± 0.14 blood cytes trol cells (10³/μl) Cases 4.20 ± 0.50 4.12 ± 0.15 4.45 ± 0.40

TABLE 3 Serum biomarkers Pre Post Rev Choles- Control 216.68 ± 3.81 223.23 ± 4.50   205.7 ± 51.90 terol Cases  219.17 ± 13.43 214.80 ± 28.01   201.5 ± 52.69 Triglic- Control 104.16 ± 5.30 148.77 ± 13.96* 117.09 ± 18.82 erides Cases  78.17 ± 3.42  100 ± 21.33  93.00 ± 22.60 Glucose Control 103.86 ± 3.50 97.32 ± 2.20  113.45 ± 10.73 Cases 101.51 ± 2.64 121.33 ± 16.18  95.33 ± 4.70 Creat- Control  0.67 ± 0.01 0.64 ± 0.01 inin Cases  0.64 ± 0.03 0.56 ± 0.02 usTnT Control    4.27 ± 0.0.32  14.39 ± 0.98** Cases  4.47 ± 0.38  11.17 ± 1.80**

Cardiac function was monitored in these patients at these three timepoints (Pre, Pst, Rev) and, in some patients, a decline in LVEF and left ventricular end-dyastolic diameter (LVEDD) to pathological levels, as defined for cardiotoxicity in the clinical guidelines, was observed (table 4).

TABLE 4 Cardiac function parameters Cardiac function parameters Pre Post Rev LVEF Control 65.86 ± 6.15 63.30 ± 7.75 63.38 ± 5.94 (%) Cases 63.31 ± 7.87  56.56 ± 12.38*   48.20 ± 8.81*** LVEDD Control 43.28 ± 0.51 42.67 ± 0.50 43.99 ± 0.58 (%) Cases 42.44 ± 2.39 46.62 ± 2.00 43.60 ± 2.25 LVEF: left ventricular ejection fraction; LVEDD: left ventricular end diastolic diameter

Among them, 10 patients that suffered cardiotoxicity during treatment defined as a decrease in 10% of FEVI below 55% (cases) were selected, and 10 additional matched patients with no affectation of cardiac function (controls). Next miRNAseq analysis using Illumina platform were performed and 200 miRNAs were detected and normalized by relative abundance.

Using three regression models, Random Forest, Negative Binomial and Elastic Net, the most differentially expressed miRNAs (n=10) between cases and controls were selected.

The random forest model achieved a cross-validated accuracy of 100% (100% sensitivity and 100% sensibility). It selected 18 miRs as the most important predictors for discriminating both groups. The Robinson and Smyth exact negative binomial test found significant differences between both groups in four miRs, three of them also present in the 18 miRs selected by random forest. Elastic net selected 16 miRs. Two of them were included in the 18 selected by the random forest model and, of those, one was also included in the 4 selected by the Robinson and Smyth test. A Venn diagram is provided to visualize the combined results of the three modeling approaches (FIG. 1). One miR has been selected by the three methods (hsa-miR-4732-3p), two have been selected by random forest and the exact negative binomial test (hsa-miR-150-5p and hsa-miR-215-5p/192-5p) and another has been selected by elastic net and random forest (hsa-miR-92b-3p). The rest of the selected miRs are the following: only selected by Robinson and Smyth exact negative binomial test: hsa-miR-148a-3p; only selected by Random Forest: hsa-miR-16-5p, hsa-miR-22-3p, 25-3p, hsa-miR-26a-5p, hsa-miR-423-5p, hsa-miR-451a, hsa-miR-486-3p/486-5p, hsa-miR-92b-3p, among others; only selected by Elastic Net: hsa-miR-30b-5p/30c-5p, among others.

Next, the expression of the 10 most significant miRNAs out of 23 miRNAs was validated (called the MirCaTox signature), in the final cohort by qPCR including the patients used in the RNAseq study. Relative values of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p were normalized to the expression of miRNA 92b-3p as described in Materials and Methods section (FIG. 2). The demographic data and clinical characteristics of the final cohort resembled the previously selected cases and controls for the RNAseq study. As expected, there was a correlation between the RNAseq and qPCR. Both techniques detected a differential expression in miRNAs defined in MirCaTox between cases and control.

Verified levels of these miRNAs were used to build a predictive model of cardiotoxic risk using a logistic mixed effects regression model. An example of algorithm using this set of miRNAs for predicting cardiotoxic risk is represented below by the following equation:

${\Pr({Cardiotoxicity})} = \frac{e^{LP}}{1 + e^{LP}}$

Where

LP=−1.228−0.041*miR4732−0.066*miR22−0.02*miR30b+0.081*miR16−0.053*miR148a+0.012*miR192−0.009*miR150p−0.055*miR215+0.0899*miR486

This model achieved an area under the curve ROC (AUC) of 0.81 (FIG. 3). This model was integrated in a software that could aid to get a probabilistic value of cardiotoxic susceptibility after input MirCaTox values for a given patient.

To investigate the biological significance of this miRNA cocktail, target genes of miRNAs included in MirCaTox were identified (FIG. 5). Among them, cardiac specific genes like TNNT2 and ACTG1 were found. These genes belong to the sarcomeric machinery of cardiac muscle cells. Accordingly, it is likely that desregulation in miRNAs included in MirCaTox signature will affect the long term function of cardiac cells.

Next, an interactome of MirCaTox, that combined direct and secondary interactions of miRNAs and proteins, was performed to identify regulated biological processes by this miRNA cocktail. Most representative pathways are shown in Table 5.

TABLE 5 Regulated biological processes in comparison of controls and cases in serum samples taken before chemotherapy. ID indicates the code number of gene ontology (GO) ID Lor Pval Name GO:0040018 0.83 0 positive regulation of multicellular organism growth GO:0010171 0.995 0 body morphogenesis GO:2000052 1.108 0.001 positive regulation of non- canonical Wnt signaling pathway GO:0043536 1.036 0.001 positive regulation of blood vessel endothelial cell migration GO:0090042 1.191 0.002 tubulin deacetylation GO:0051123 0.747 0.002 RNA polymerase II transcriptional preinitiation complex assembly GO:0072202 1.235 0.003 cell differentiation involved in metanephros development GO:0070897 0.679 0.003 DNA-templated transcriptional preinitiation complex assembly GO:0061005 0.754 0.003 cell differentiation involved in kidney development GO:0060271 0.301 0.003 cilium morphogenesis GO:0014902 0.468 0.003 myotube differentiation GO:0010165 0.673 0.003 response to X-ray GO:0006261 0.356 0.004 DNA-dependent DNA replication GO:0035116 0.971 0.004 embryonic hindlimb morphogenesis GO:0006790 0.367 0.004 sulfur compound metabolic process GO:0033262 1.064 0.004 regulation of nuclear cell cycle DNA replication GO:0030195 0.883 0.004 negative regulation of blood coagulation GO:0050819 0.883 0.004 negative regulation of coagulation GO:1900047 0.883 0.004 negative regulation of hemostasis GO:0072089 0.388 0.004 stem cell proliferation GO:0060325 0.876 0.004 face morphogenesis GO:0060242 1.122 0.005 contact inhibition GO:0001656 0.549 0.005 metanephros development GO:0006266 0.814 0.005 DNA ligation GO:0072659 0.298 0.005 protein localization to plasma membrane GO:0048854 0.752 0.005 brain morphogenesis GO:0006893 0.601 0.005 Golgi to plasma membrane transport GO:0019985 0.571 0.005 translesion synthesis GO:0043001 0.733 0.006 Golgi to plasma membrane protein transport GO:0032535 0.256 0.006 regulation of cellular component size GO:0033260 0.802 0.006 nuclear cell cycle DNA replication GO:0046112 0.848 0.006 nucleobase biosynthetic process GO:0044272 0.474 0.007 sulfur compound biosynthetic process GO:0090002 0.32 0.009 establishment of protein localization to plasma membrane GO:0035137 0.808 0.009 hindlimb morphogenesis GO:0060020 0.984 0.009 Bergmann glial cell differentiation GO:0051188 0.432 0.009 cofactor biosynthetic process GO:0050818 0.484 0.009 regulation of coagulation GO:0021697 0.796 0.01 cerebellar cortex formation GO:0051291 −0.335 0.01 protein heterooligomerization GO:0045815 −0.353 0.01 positive regulation of gene expression, epigenetic GO:0006323 −0.254 0.009 DNA packaging GO:0002244 −0.328 0.009 hematopoietic progenitor cell differentiation GO:0043201 −1.041 0.009 response to leucine GO:0050728 −0.491 0.008 negative regulation of inflammatory response GO:2000756 −0.482 0.008 regulation of peptidyl-lysine acetylation GO:0032438 −0.984 0.008 melanosome organization GO:0009749 −0.351 0.008 response to glucose GO:0072576 −0.888 0.008 liver morphogenesis GO:0071103 −0.217 0.007 DNA conformation change GO:0019083 −0.241 0.007 viral transcription GO:0006342 −0.324 0.007 chromatin silencing GO:0006336 −0.491 0.007 DNA replication-independent nucleosome assembly GO:0055075 −1.073 0.007 potassium ion homeostasis GO:0019080 −0.235 0.007 viral gene expression GO:0031050 −0.625 0.007 dsRNA fragmentation GO:0070918 −0.625 0.007 production of small RNA involved in gene silencing by RNA GO:0040016 −1.025 0.007 embryonic cleavage GO:0009746 −0.353 0.007 response to hexose GO:0034724 −0.487 0.007 DNA replication-independent nucleosome organization GO:0002440 −0.38 0.007 production of molecular mediator of immune response GO:0000338 −0.892 0.006 protein deneddylation GO:1903307 −1.008 0.006 positive regulation of regulated secretory pathway GO:0006414 −0.279 0.006 translational elongation GO:0001974 −0.723 0.006 blood vessel remodeling GO:0002700 −0.481 0.006 regulation of production of molecular mediator of immune response GO:0006406 −0.353 0.006 mRNA export from nucleus GO:0065005 −1.067 0.005 protein-lipid complex assembly GO:0006497 −0.549 0.005 protein lipidation GO:0002697 −0.271 0.005 regulation of immune effector process GO:0019058 −0.181 0.005 viral life cycle GO:0044033 −0.234 0.005 multi-organism metabolic process GO:0000183 −0.619 0.005 chromatin silencing at rDNA GO:0050434 −0.539 0.005 positive regulation of viral transcription GO:0043486 −0.524 0.004 histone Exchange GO:0002706 −0.468 0.004 regulation of lymphocyte mediated immunity GO:0048385 −1.084 0.004 regulation of retinoic acid receptor signaling pathway GO:0035196 −0.686 0.004 production of miRNAs involved in gene silencing by miRNA GO:0045637 −0.319 0.004 regulation of myeloid cell differentiation GO:0043902 −0.37 0.004 positive regulation of multi- organism process GO:1901533 −0.902 0.003 negative regulation of hematopoietic progenitor cell differentiation GO:0042158 −0.546 0.003 lipoprotein biosynthetic process GO:0045653 −1.357 0.003 negative regulation of megakaryocyte differentiation GO:0048771 −0.43 0.003 tissue remodeling GO:0016311 −0.234 0.003 Dephosphorylation GO:0071604 −1.018 0.003 transforming growth factor beta production GO:0071634 −1.018 0.003 regulation of transforming growth factor beta production GO:0042541 −1.057 0.003 hemoglobin biosynthetic process GO:0020027 −0.984 0.003 hemoglobin metabolic process GO:0002703 −0.415 0.003 regulation of leukocyte mediated immunity GO:0071705 −0.205 0.003 nitrogen compound transport GO:0031055 −0.601 0.003 chromatin remodeling at centromere GO:0051028 −0.333 0.003 mRNA transport GO:0006417 −0.247 0.003 regulation of translation GO:0034284 −0.381 0.002 response to monosaccharide GO:0009743 −0.36 0.002 response to carbohydrate GO:0016233 −0.707 0.002 telomere capping GO:0048524 −0.407 0.002 positive regulation of viral process GO:1901532 −0.637 0.002 regulation of hematopoietic progenitor cell differentiation GO:0034080 −0.667 0.002 CENP-A containing nucleosome assembly GO:0061641 −0.667 0.002 CENP-A containing chromatin organization GO:0045638 −0.524 0.002 negative regulation of myeloid cell differentiation GO:0051168 −0.322 0.001 nuclear export GO:0031348 −0.493 0.001 negative regulation of defense response GO:0045652 −0.974 0.001 regulation of megakaryocyte differentiation GO:0044346 −1.095 0.001 fibroblast apoptotic process GO:0050657 −0.343 0.001 nucleic acid transport GO:0050658 −0.343 0.001 RNA transport GO:0051236 −0.343 0.001 establishment of RNA localization GO:0032906 −1.146 0.001 transforming growth factor beta2 production GO:0032909 −1.146 0.001 regulation of transforming growth factor beta2 production GO:0046784 −1.185 0.001 viral mRNA export from host cell nucleus GO:0051290 −0.903 0.001 protein heterotetramerization GO:0015931 −0.345 0.001 nucleobase-containing compound transport GO:0006403 −0.343 0 RNA localization GO:0030219 −0.716 0 megakaryocyte differentiation GO:0006405 −0.425 0 RNA export from nucleus GO:0006413 −0.302 0 translational initiation GO:0006987 −1.285 0 activation of signaling protein activity involved in unfolded protein response GO:0006335 −1.156 0 DNA replication-dependent nucleosome assembly GO:0034723 −1.156 0 DNA replication-dependent nucleosome organization GO:0010804 −1.127 0 negative regulation of tumor necrosis factor-mediated signaling pathway GO:0006446 −0.587 0 regulation of translational initiation GO:2000269 −1.351 0 regulation of fibroblast apoptotic process GO:0016458 −0.349 0 gene silencing GO:0031047 −0.526 0 gene silencing by RNA

Significant biological processes are listed by P value (pval). Lor positive value is indicative of increased levels of miRNAs with the consequent repression of biological processes whereas Lor negative value is indicative of decreased levels of miRNA what would result in desrepresion of target genes in cases versus controls. Thus, a repression of Wnt signaling pathway, a known pathway involved in heart development, together with repression of biological processess related to blood vessel development, endothelial cell migration, embryonic hind limb morphogenesis, myotube differentiation and sulphur compound metabolic process, were observed, what are also related to cardioprotection. The ERK1/2 signalling pathway is involved in sulphur dioxide preconditioning-induced protection against cardiac dysfunction in isolated perfused rat heart subjected to myocardial ischemia/reperfusion (Huang P et al. Int J Mol Sci. (2013); Hydrogen sulphide attenuates cardiac dysfunction in a rat model of heart failure: a mechanism through cardiac mitochondrial protection. Wang X et al. Biosci Rep. (2011); “Hydrogen sulfide attenuates cardiac dysfunction in a rat model of heart failure: a mechanism through cardiac mitochondrial protection. Wang X, et al. Biosci Rep. 2011 April; 31(2):87-98; Garcia NA1, Moncayo-Arlandi J1,2, Vazquez A1, Genovés P1, Calvo CJ3, Millet J3, Marti N4,5, Aguado C4,5, Knecht E4,5, Valiente-Alandi I6, Montero JA1, Díez-Juan A7,8, Sepúlveda P. hydrogen sulfide improves cardiomyocyte function in a cardiac arrest model. Ann Transplant. (2017) May 9; 22:285-295). Moreover, this miRNA combination was related to increase in coagulation and hemostasis. In contrast, biological processes like translational initiation, nuclear export, RNA localization, response to carbohydrate, tissue remodelling, transforming growth factor beta production and regulation of tumor necrosis factor-mediated signalling pathway and fibroblast apoptosis were unrepressed.

Assays in Cardiomyocytes

The specific signature of miRNAs has been detected in serum. Since the pathways related to miRNAs are linked to cardiomyocytes, the copy number of the miRNAs of interest in human cardiomyocytes was also measured. As observed in FIG. 4, the miRNAs previously detected in serum were also present in human cardiomyocytes (left panels) and their expression level is altered when cells are damaged by Doxorubicin. Moreover, the effect of Doxorubicin on cardiac cells resulted in similar modulation than the variation observed in patients. As shown in the right panel of FIG. 4, the copy number of miRNA-22-3p increases both in cells and serum of patients after anthracycline treatment whereas miRNA-30b, miRNA-148a, miRNA-150-5p, and miRNA-4732-3p decrease their expression levels. These results enforce the usefulness of the signature of miRNAs presented in this patent as sensor of cardiac wellness. Indeed, this set of miRNAs regulates genes involved in the aforementioned biological pathways (FIG. 5)

Finally, we wanted to see if modulation of miRNA levels could result in cardioprotection of cardiomyocyte cells in culture. For this purpose, we transfected NRCMs with miRNA mimics and evaluated their cardioprotective effect in terms of increase in cell viability and decrease in apoptosis in comparison to non-treated cultures after doxorubicin treatment (FIG. 6). As observed, miR-30b-5p, miR-150-5p and miR-4732-3p protected NRVCM against doxorrubicin treatment. To test if cardioprotection could be extended to other types of injury we set up an in vitro experiment where cells were submmited to ischemia followed by reperfusion (I/R) as explained in materials and methods. Since reoxygenation induce both cell survival and apoptosis, we observed a strong upregulation of miR-150-5p and miR-4732 (FIG. 7), indicating that therapies aiming to modulate MirCaTox miRNAs could result in therapeutic targets to prevent cardiotoxicity. Moreover, when we performed overexpression of miR-150-5p or miR-4732-3p in NRVCM and subjected them to I/R, transfected cells showed reduced levels of ROS and apoptosis (FIG. 8). 

1. Use of a set of 10 circulating miRNAs, consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, as biomarker of prediction of cardiotoxicity risk in cancer patients susceptible to receive anthracyclines chemotherapy.
 2. A method for predicting cardiotoxicity risk in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy comprising: i. Determining the expression levels of a combination of 10 circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, in a biological sample isolated from the patient, and ii. Comparing the expression levels determined in i) with the expression levels of said miRNAs from a biological sample isolated from a control subject not suffering from cardiotoxicity, wherein the alteration of the expression of these miRNAs, comparing with the expression of said miRNAs in a control subject, is indicative of cardiotoxicity risk.
 3. A method for predicting the cardiotoxicity risk probability in a cancer patient receiving, or susceptible to receive, anthracyclines chemotherapy comprising: a. Determining the expression levels of a combination of 10 circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p, in a biological sample isolated from the patient, and b. Introducing the expression data obtained in a), in the following formula: ${\Pr({Cardiotoxicity})} = \frac{e^{LP}}{1 + e^{LP}}$ wherein, LP=−1.228−0.041*miR4732−0.066*miR22−0.02*miR30b+0.081*miR16−0.053*miR148a+0.012*miR192−0.009*miR150p−0.055*miR215+0.0899*miR486 and wherein the obtained result is the expected probability of cardiotoxicity (Pr(cardiotoxicity)).
 4. Method according to claim 1, wherein the biological sample is a liquid biopsy.
 5. Method according to claim 4, wherein the liquid biopsy is serum.
 6. Method according to claim 1, wherein the patient is a breast cancer patient.
 7. A method for the prevention of cardiotoxicity in patients receiving or susceptible to receive anthracyclines chemotherapy that comprises modulating the expression levels of the set of 10 circulating miRNAs consisting of miRNA 16-5p, miRNA 22-3p, miRNA 30b-5p/30c-5p, miRNA 92b-3p, miRNA 148a-3p, miRNA-150-5p, miRNA-192-5p, miRNA 215-5p, miRNA 486-3p/486-5p and miRNA-4732-3p. 